Project Summary ? Project 1 Over the last four years of P50 funding to the VCU Alcohol Research Center (VCU ARC), our laboratory used Diversity Outbred (DO) mouse mice from Jackson Laboratories (http://do.jax.org) for behavioral genetics and initial genomic studies on progressive ethanol consumption. DO mice originate from 8 progenitor mouse strains chosen to maximize genetic diversity and utilize a breeding scheme producing a high degree of heterozygosity for fine mapping complex traits such as ethanol consumption. As such, DO mice more faithfully mimic genetic aspects seen with alcohol use disorder (AUD). As expected, based upon preliminary work in DO progenitor strains, behavioral studies using a progressive ethanol consumption model (intermittent ethanol access, IEA) on over 600 DO mice showed a broad distribution of consumption values (~0.5?38 g/kg/24h) that shifted to significantly higher intake over the 4 week experiment. Genotyping identified 10 genome-wide significant or suggestive QTL with LOD ? 6 and support intervals generally < 2 Mb. Strikingly, quantitative trait loci (QTL) for the first week of drinking differed from those during the last week of consumption. Ongoing haplotype analysis and integration of RNAseq data from prefrontal cortex have identified provisional candidate genes, including two that have been implicated in human genome-wide association studies on alcohol consumption or dependence. We hypothesize in this renewal that extension of this DO behavioral QTL and genomic data will identify novel candidate genes and gene networks contributing to ethanol consumption behaviors in mice and that such data will inform existing and future human genetic studies and therapeutic efforts on AUD. Our specific aims thus describe: 1) Further expression genetics analysis in nucleus accumbens, allele specific expression analysis, and chromatin 3D conformation analyses to identify and refine a list of positional candidate genes for behavioral QTL associated with initial ethanol intake vs. progressive consumption; 2) Gene network analysis of RNAseq data in both prefrontal cortex and nucleus accumbens across 200 DO mice to identify networks and possible mechanisms tightly associated with consumption differing between first and last week; and 3) Validation of candidate genes or network hubs as functioning in ethanol behaviors, including initial or progressive ethanol consumption, using invertebrate models (collaboration with Projects 2 and 3 of this Center proposal), rodent models (this project and Rodent Behavioral Core). Further, the work of this project will inform and be informed by novel human genetic studies described in Projects 4 and 5. Throughout this project, the VCU ARC Bioinformatics and Analysis Core will provide critical support for analysis of RNAseq data and Capture-C chromatin conformation studies, and the choice of candidate genes and networks. This novel gene discovery and network analysis effort will have major interactions with all other components of VCU ARC and will inform the field of alcohol research with understanding of mechanisms involved in the transition to abusive drinking, and candidate genes/mechanisms for potential targeting by future therapeutic efforts.